應用線狀結構照明提升雙光子顯微鏡解析度

Resolution Enhancement in Two-photon Microscopy by Applying Structured Line Illumination

林祺登(Chi-deng Lin)

指導教授 ： 陳思妤

國立中央大學/理學院/光電科學與工程學系/碩士(2015年)

摘要

本研究主要為改良雙光子螢光顯微鏡(Two-Photon Microscopy, TPM)，應用線狀光形的干涉條紋當作結構照明(Structured Illumination)以提升解析度。結構照明顯微鏡(Structured Illumination Microscopy)可將頻譜空間拓展至傳統顯微鏡的兩倍，並使解析度提升至傳統顯微鏡的兩倍。而雙光子螢光與激發光強度成平方關係的特點，使雙光子螢光顯微鏡有良好的光學切片能力，可以改善結構照明顯微鏡縱向解析度的不足，且雙光子螢光之結構照明可提升影像的空間頻率為將近原本的三倍。因此可透過此系統架構，不但使解析度提高亦可以擁有光學切片能力。
本研究可分為實驗與模擬兩部分。在模擬方面，依照實驗架構的規格，模擬雙光子螢光顯微鏡以線狀結構照明激發的影像，並將影像經由演算法重建還原，其解析度約提升2.57倍；而在實驗部分，成功架設出應用線狀結構照明之雙光子螢光顯微鏡，以量子點奈米微粒(Quantum Dot Nanosphere)作為空間解析度量測的樣本，並經由演算法將影像重建還原，可得其橫向空間解析度約提升至1.7倍。

關鍵字

雙光子螢光 ； 結構照明顯微鏡 ； 線狀結構照明

並列摘要

The aim of this study is to enhance the spatial resolution in two-photon microscopy (TPM) by applying a line-shaped structured illumination. Since structured illumination microscopy (SIM) expands the effective frequency spectrum space twice the size bigger of a traditional microscopy’s, its spatial resolution is therefore two times better. Combining it with TPM’s optical sectioning ability, which is due to the relation between the emission and excitation intensity, we can increase the vertical spatial resolution, and also raise the image’s spatial resolution up nearly three times better. Therefore, utilizing our system not only improves the lateral resolution, but also provides optical sectioning ability.
This study is separated into simulation and experimental parts. In the simulation part, by using the parameters shown in the spec of the experiment setup, we simulate the whole process of a TPM image being excited by a structured line illumination, and then use the results to reconstruct the image via algorithm, in which, we were able to come up with a improve factor of 2.57. Later in the experimental part, by using quantum dot nanospheres as our samples for lateral resolution measurement purposes, and exciting two-photon fluorescence while applying structured line illumination, we successfully reconstructed the image via algorithm with a lateral resolution improve factor of 1.7. Finally, we compare the experiment results with the simulation, and discuss its advantages.

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